A wide variety of lipases of microbial origin (both intracellular and extracellular), as well as plant and animal origin are known. For a general discussion of extracellular microbial lipases, see A. R. Macrae, p. 225ff in Microbial Enzymes and Biotechnology (Ed. W. Fogarty), ISBN 0-85334-185-0, Applied Science Publishers Ltd., 1983.
Non-specific lipases from the following microorganisms are known: Staphylococcus aureus (Vadehra, D. V. (1974). Lipids, 9, 158), Penicillium cyclopium (Okumura, S., et al. (1976). Agricultural and Biological Chemistry, 40, 655 and Renshaw E. C. and San Clemente C. L. (1966) Developments in Industrial Microbiology, 8, 214), Corynebacterium acnes (Hassing, G. S. (1971). Biochimica et Biophysica Acta, 242, 381 and Pablo G. (1974) The Journal of Investigative Dermatology, 63, 231), Propionibacterium acnes (Ingham, E. et al. (1981). Journal of General Microbiology, 124, 393), Candida cylindracea (also known as C. rugosa) (Benzonana, G. & Esposito, S. (1971). Biochimica et Biophysica Acta, 231, 15; and Kimura Y. (1983) Eur. J. Appl. Microbiol. Biotechnol., 17, 107), Candida curvata (D. Montet et al. (1985), Fette Seifen Anstrichmittel, 87, 181). However, data in the literature referred to and in an example of this specification demonstrate that all these lipases have insufficient thermostability for long-term use at about 60.degree. C. or higher. Also S. aureus, C. acnes and P. acnes are suspected or proven pathogens.
Lipase from Geotrichum candidum (Jensen, R. G. (1974) Lipids, 9, 149; Jensen, R. G. et al. (1972) Lipids, 7, 738; and Tsujisaka, Y. and Iwai, M. (1984) Kagaku to Kogyo, 58, 60) is positionally non-specific, but is highly specific for certain unsaturated fatty acyl groups. Further, it is not thermostable.
Further, lipases from Humicola lanuginosa (Liu, W. H., Beppu, T. & Arima, K. (1973). Agricultural and Biological Chemistry, 37, 1349) and Chromobacterium viscosum (Sugiura, M. & Isobe, M. (1975). Chemical and Pharmaceutical Bulletin, 23, 1226) have been described as non-specific. However, later results (Biotechnology and Genetic Engineering Reviews, Vol. 3 (Sep. 1985), page 200) show that these two lipases are, in fact, specific. Data in an example of this specification also demonstrate that the C. viscosum lipase is specific.
Immobilized non-specific lipase is disclosed in Y. Kimura et al., Eur. J. Microbiol. Biotechnol. 17 (1983), 107-112. The lipase is derived from Candida cylindracea, and the data in the article show that the immobilized lipase has optimum temperature about 40.degree. C., and that there is significant deactivation at 50.degree. C.
Immobilized non-specific lipase and its use for random interesterification of fat are described in Macrae, A. R. (1983), Journal of the American Oil Chemists' Society (JAOCS), 60, 291. However, the process temperature was only 40.degree. C. This low temperature was probably chosen due to the poor thermostability of the Candida cylindracea lipase.
There is a need for thermostable, non-specific lipase for processing high-melting substrates at about 60.degree. C. or higher without solvent, e.g. for randomization of fat in the margarine industry. Reference is made to A. R. Macrae and R. C. Hammond: "Present and Future Applications of Lipases", Biotechnology and Genetic Engineering Reviews, 3, 193-217 (1985). Prior-art preparations are not sufficiently heat-stable, and it is the object of the invention to provide non-specific lipase that is thermostable enough for long-term use at 60.degree. C. or higher in soluble or immobilized form. The lipase should be microbial, as these can be produced economically.